DNA microarrays have been developed as tools for high throughput analysis of gene expression, but despite their important role in biology and medicine, levels of gene expression do not always correlate with levels of protein expression and concentration. Protein and peptide arrays have been designed to directly measure protein concentration and protein: protein interactions but their usefulness presently is limited due to the limited diversity of capture molecules- only a few thousand- that can be immobilized on the array. These limitations stem primarily from the lack of adequate reagents and the inability to immobilize them on the arrays in a sufficiently dense format. We are proposing to develop novel arrays, in which hundreds of thousands of combinatorially-generated peptides, synthesized on chromatography resin beads, are immobilized on a glass or plastic surfaces. This will provide arrays with orders of magnitude more diversity than standard arrays, with the peptides present in three-dimensions to promote protein binding. We will extend our preliminary research demonstrating the feasibility of this concept by: a) increasing the number of peptides that can be immobilized in an array by more than 10-fold to > 250,000; b) optimizing the Pathway BioImager instrument to expedite the array reading; c) optimizing handling conditions; d) evaluating and minimizing false positive and false negative rates using standardized reagents and plasma samples; e) developing alternate methods for ligand sequencing. Upon successful completion of this Phase 1 proposal we will submit a Phase 2 application in which we propose to extend the use of the arrays to identification of melanoma biomarkers using 200 patient samples obtained from John Kirkwood, MD (U Pittsburgh Cancer Center). We also will explore additional methods of in situ sequencing of the beads in the arrays, which will allow us to expand the diversity of peptides in the array to > 1,000,000. Finally, we will complete development, manufacturing and commercialization of the array for research only use by partnering with an established scientific instrument maker while preserving commercial applications within Prolias for out-licensing opportunities. The proposal describes the development and optimization of novel, highly diverse peptide microarrays. These arrays are produced by immobilizing hundreds of thousands of peptides, synthesized on chromatography resin beads, onto glass slides. These arrays ultimately will be used to analyze samples, including autoantibodies, from melanoma patients to discover new biomarkers for disease diagnosis, prognosis, and evaluation of treatment. [unreadable] [unreadable] [unreadable]